SCAPE Microscopy with Phase Modulating Element and Image Reconstruction

ABSTRACT

A scanning element routes a sheet or beam of excitation light through a first set of optical components and into a sample at an oblique angle. The position of the excitation light within the sample varies depending on the orientation of the scanning element. Fluorophores within the sample emit fluorescent detection light. The first set of optical components route the detection light back to the scanning element, and the scanning element routes the detection light through a second set of optical components and into a camera. The second set of optical components includes a phase modulating element that induces a controlled aberration so as to homogenize point spread functions of points at, above, and below a focal plane when measured at the camera. In some embodiments, the images captured by the camera are processed to correct for aberration by performing deconvolution of a point spread function.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of U.S. Provisional Application62/343,112, filed May 30, 2016, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This invention is made with government support from the NIH under GrantNos. 5U01NS094296-01, 1R01NS076628, 1R01NS063226, and R21NS053684; NSFgrants CBET-0954796 and IGERT 0801530; and DOD grant MURIW911NF-12-1-0594. The Government has certain rights in the invention.

BACKGROUND

SCAPE microscopy is a technique for high-speed 3D microscopy that usesswept, confocally aligned planar excitation. Some examples of SCAPEimaging systems are disclosed in publication WO 2015/109323, which isincorporated herein by reference in its entirety. In SCAPE, an obliquesheet of light (e.g., laser light) is swept through the sample, andfluorescence from the sample is captured by a camera to produce animage. SCAPE can achieve resolutions rivaling techniques such aslight-sheet, confocal, and two-photon microscopy, but can operate atmuch higher speeds.

In SCAPE systems, the excitation path includes the optical componentsstarting at the light source and ending at the sample; and the detectionpath includes the optical components starting at the sample and endingat the camera. Some embodiments of SCAPE microscopy use two objectivelenses in the detection arm placed at an angle with respect to oneanother for image rotation. But this configuration results in a net lossof light captured at the detector, which can adversely affect thenumerical aperture, resolution, and light throughput of the imagingsystem.

SUMMARY OF THE INVENTION

One aspect of the invention is directed to a first apparatus thatcomprises a first set of optical components having a proximal end and adistal end, the first set of optical components including an objectivedisposed at the distal end of the first set of optical components. Thisapparatus also comprises a second set of optical components having aproximal end and a distal end, the second set of optical componentsincluding a phase modulating element. This apparatus also comprises ascanning element that is disposed proximally with respect to theproximal end of the first set of optical components and proximally withrespect to the proximal end of the second set of optical components. Thescanning element is arranged to route excitation light arriving at thescanning element so that the excitation light will pass through thefirst set of optical components in a proximal to distal direction andproject into a sample that is positioned distally beyond the objective.The excitation light is projected into the sample at an oblique angle,and the excitation light is projected into the sample at a position thatvaries depending on an orientation of the scanning element. The firstset of optical components is arranged to route detection light from thesample in a distal to proximal direction back to the scanning element.The scanning element is also arranged to route the detection light sothat the detection light will pass through the second set of opticalcomponents in a proximal to distal direction. This apparatus alsocomprises a camera optically positioned to capture images formed by thedetection light that has passed through the second set of opticalcomponents. The phase modulating element induces a controlled aberrationso as to homogenize point spread functions of points at, above, andbelow a focal plane when measured at the camera.

In some embodiments of the first apparatus, the phase modulating elementcomprises a phase plate. In some embodiments of the first apparatus, thephase modulating element comprises a spatial light modulator. In someembodiments of the first apparatus, the phase modulating elementcomprises a deformable mirror.

In some embodiments of the first apparatus, the objective has a pupilplane, and the phase modulating element is positioned at a plane that isconjugate to the pupil plane of the objective. Some of these embodimentsfurther comprise an aperture stop positioned (a) adjacent to the phasemodulating element and (b) proximally with respect to the phasemodulating element.

In some embodiments of the first apparatus, the detection light forms astationary conjugate image plane between the proximal end and the distalend of the second set of optical components.

In some embodiments of the first apparatus, the light detector arraycomprises a 2D image sensor, and the camera sequentially captures aplurality of images formed by the detection light that has passedthrough the second set of optical components, each of the plurality ofimages corresponding to a respective frame of image date. In theseembodiments, the apparatus further comprises a processor programmed tocorrect for aberration in the frames of image data. In some of theseembodiments, the processor corrects for aberration in the frames ofimage data by performing deconvolution of a point spread function. Insome of these embodiments, the point spread function is either ameasured point spread function or a simulated point spread function.

In some embodiments of the first apparatus, the light detector arraycomprises a linear image sensor. In some of these embodiments, thescanning element comprises an x-y galvanometer. In some of theseembodiments, the excitation light has a wavelength such that excitationof a fluorophore in the sample requires near-simultaneous absorption ofa plurality of photons.

Some embodiments of the first apparatus further comprise a beam splitterdisposed between the proximal end of the second set of opticalcomponents and the scanning element, and a source of the excitationlight. In these embodiments, the source of the excitation light is aimedso the excitation light is directed into the beam splitter, the beamsplitter is arranged to route the excitation light towards the scanningelement, and the beam splitter is arranged to route detection lightarriving from the scanning element into the proximal end of the secondset of optical components.

In some embodiments of the first apparatus, the excitation lightcomprises a sheet of excitation light. This sheet of excitation lightmay be generated by at least one of (a) a cylindrical lens arranged toexpand light from a light source into the sheet of excitation light; (b)an aspheric mirror arranged to expand light from a light source into thesheet of excitation light; (c) a spatial light modulator arranged toexpand light from a light source into the sheet of excitation light; (d)a second scanning element arranged to expand light from a light sourceinto the sheet of excitation light; and (e) an oscillating galvanometermirror arranged to expand light from a light source into the sheet ofexcitation light.

Some embodiments of the first apparatus further comprise a beam splitterdisposed between the proximal end of the second set of opticalcomponents and the scanning element, and a source of the excitationlight. In these embodiments, the source of the excitation light is aimedso the excitation light is directed into the beam splitter, the beamsplitter is arranged to route the excitation light towards the scanningelement, the beam splitter is arranged to route detection light arrivingfrom the scanning element into the proximal end of the second set ofoptical components, the light detector array comprises a 2D imagesensor, the excitation light comprises a sheet of excitation light, thecamera sequentially captures a plurality of images formed by thedetection light that has passed through the second set of opticalcomponents, each of the plurality of images corresponding to arespective frame of image date, and the apparatus further comprises aprocessor programmed to correct for aberration in the frames of imagedata. In some of these embodiments, the processor corrects foraberration in the frames of image data by performing deconvolution of apoint spread function. In some of these embodiments, the objective has apupil plane, and the phase modulating element is positioned at a planethat is conjugate to the pupil plane of the objective. In some of theseembodiments, the phase modulating element has a phase profile of

θ(x, y)=2πα(x ³ +y ³)

where x and y are normalized pupil coordinates and α is a tuningparameter set to |Ψ/2|, where

Ψ(u ₂ , v ₂ ; dz)=−(1/2λ)*NA ²*(u ₂ ² +v ₂ ²)*dz/n,

where λ is a wavelength of light passing through the phase modulatingelement, NA is a numerical aperture of the objective, u and v arenormalized pupil coordinates, n is an index of refraction of animmersion medium underneath the objective, and dz is one half of a rangeof depths to be imaged.

In some embodiments of the first apparatus, the phase modulating elementhas a phase profile of

θ(x, y)=2πα(x ³ +y ³)

where x and y are normalized pupil coordinates and a is a tuningparameter set to |Ψ/2|, where

Ψ(u ₂ , v ₂ ; dz)=−(1/2λ)*NA ²*(u ₂ ² +v ₂ ²)*dz/n,

where λ is a wavelength of light passing through the phase modulatingelement, NA is a numerical aperture of the objective, u and v arenormalized pupil coordinates, n is an index of refraction of animmersion medium underneath the objective, and dz is one half of a rangeof depths to be imaged.

Another aspect of the invention is directed to a second apparatus thatcomprises an objective and a plurality of optical elements disposed in adetection path arranged to receive light from the objective. Theplurality of optical elements disposed in the detection path includes ascanning element. The scanning element is arranged to (a) routeexcitation light into the objective so as to generate a sweepingexcitation beam through a forward image plane of the objective and (b)simultaneously route image light returning through the objective alongthe detection path to form a conjugate image. This apparatus furthercomprises a light detector array positioned to capture images of theconjugate image, and a phase modulating element disposed in thedetection path between the scanning element and the light detectorarray. The phase modulating element extends a depth of field in thedetection path.

In some embodiments of the second apparatus, the phase modulatingelement extends the depth of field by inducing a controlled aberrationso as to homogenize point spread functions of points at, above, andbelow a focal plane when measured at the light detector array.

In some embodiments of the second apparatus, the aberration is correctedby an image processing algorithm. In some embodiments of the secondapparatus, the aberration is corrected by deconvolution of a measured orsimulated point spread function. In some embodiments of the secondapparatus, the light detector array comprises a 2D image sensor. In someembodiments of the second apparatus, the light detector array comprisesa linear image sensor.

Another aspect of the invention is directed to a third apparatus thatcomprises a light source; a cylindrical lens or a scanner that expandslight from the light source into a sheet of light; a beam splitterdisposed in a path of the sheet of light; a scanning element disposed ina path of the sheet of light; a first telescope having a proximal endand a distal end, with an objective disposed at the distal end of thefirst telescope; and a second telescope having a proximal end and adistal end, the second telescope having an optical axis. The beamsplitter routes the sheet of light towards the scanning element. Thescanning element routes the sheet of light into the proximal end of thefirst telescope. The first telescope routes the sheet of light in aproximal to distal direction through the objective, accepts fluorescentlight through the objective and routes the fluorescent light in a distalto proximal direction back to the scanning element. The scanning elementroutes the fluorescent light through the beam splitter and into theproximal end of the second telescope. The second telescope forms animage from the fluorescent light at a conjugate image plane. Thisapparatus further comprises a camera positioned on the same optical axisas the second telescope and configured to capture blurred images of theconjugate image plane; a phase modulating element disposed between thesecond telescope and the camera; and an image processor programmed todeblur images captured by the camera.

In some embodiments of the third apparatus, the light source comprises alaser. In some embodiments of the third apparatus, the phase modulatingelement comprises a phase plate. In some embodiments of the thirdapparatus, the phase modulating element comprises at least one of aspatial light modulator and a deformable mirror. In some embodiments ofthe third apparatus, the image processor is programmed to deblur theimages using a deconvolution algorithm.

Another aspect of the invention is directed to a first method of imaginga sample. This method comprises projecting a sheet of excitation lightinto a sample, wherein the sheet of excitation light is projected intothe sample at an oblique angle, and wherein the sheet of excitationlight is projected into the sample at a position that varies dependingon an orientation of a scanning element. This method also comprisesrouting detection light arriving from the sample back to the scanningelement, and using the scanning element to reroute the detection lightinto an optical system that induces a controlled aberration so as tohomogenize point spread functions of points at, above, and below a focalplane when measured at the camera. This method also comprises using theaberrated detection light to form a plurality of images at a pluralityof times, each of the times corresponding to a different orientation ofthe scanning element, and capturing the plurality of images.

Some embodiments of the first method further comprise correcting for theaberration in the plurality of images.

In some embodiments of the first method, the correcting step comprisesperforming deconvolution of a point spread function. In some embodimentsof the first method, the point spread function is either a measuredpoint spread function or a simulated point spread function.

In some embodiments of the first method, the aberration is induced by aphase modulating element having a phase profile of

θ(x, y)=2πα(x ³ +y ³)

where x and y are normalized pupil coordinates and a is a tuningparameter set to |Ψ/2|, where

Ψ(u ₂ , v ₂ ; dz)=−(1/2λ)*NA ²*(u ₂ ² +v ₂ ²)*dz/n,

where λ is a wavelength of light passing through the phase modulatingelement, NA is a numerical aperture of the objective, u and v arenormalized pupil coordinates, n is an index of refraction of animmersion medium underneath the objective, and dz is one half of a rangeof depths to be imaged.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an embodiment of a SCAPE microscope that uses a phasemodulating element.

FIG. 2A depicts a design for forming a sheet of light in which allpositions on the sheet are illuminated simultaneously.

FIG. 2B depicts a design for rapidly scanning a pencil beam of light soas to form a virtual sheet of light.

FIGS. 3A and 3B depict, respectively, a phase function and acorresponding irradiance point spread function.

FIGS. 4A and 4B depict the sectioning effect of the oblique light sheetused in SCAPE microscopy.

FIG. 5 depicts an embodiment of a SCAPE microscope that uses areflective phase modulating element.

FIG. 6 depicts an embodiment of a SCAPE microscope that uses a phasemodulating element, a scanning element with two degrees of freedom, anda linear sensor.

Embodiments will hereinafter be described in detail below with referenceto the accompanying drawings, wherein like reference numerals representlike elements.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This application describes an alternative approach for implementingSCAPE microscopy that avoids many of the problems associated with thoseembodiments of SCAPE that use two objective lenses in the detection armplaced at an angle with respect to one another for image rotation. Thisalternative approach relies on a phase modulating element to extend theeffective depth of field of the detection arm.

FIG. 1 depicts a first embodiment of a SCAPE microscope that uses aphase modulating element (PME) 170. Examples of phase modulatingelements that may be used in this embodiment include but are not limitedto phase plates (e.g., cubic, logarithmic, etc.) and spatial lightmodulators (SLMs).

A beam of light from a light source (e.g., laser 100 or an LED) having awavelength within the excitation spectrum of a fluorophore of interestis passed through sheet forming optics 110. This sheet forming optics110 converts the beam into a sheet of light and also dictates thegeometric properties of the light that will excite the fluorescence inthe sample.

In some embodiments, the sheet forming optics 110 shapes the light fromthe light source 100 into a true sheet of light (i.e., a sheet in whichall positions on the sheet of light are illuminated simultaneously). Oneapproach for accomplishing this is depicted in FIG. 2A. A light source(e.g., laser 100 or an LED) emits light (e.g., monochromatic, coherent,polarized light; or incoherent light), and that light is directedthrough an isotropic beam expander 201, 202 that uniformly magnifies theprofile of the light source. From there, the light passes through ananamorphic beam expander that magnifies beam from a circular shape to anelliptical shape. This anamorphic beam expander may be implemented, forexample, using a pair of cylindrical lenses 211, 212 and/or matchedprism pairs and/or aperture cropping with a slit, etc.

The light is then reflected off of a sliding mirror 220 and is focusedalong the expanded dimension by a third cylindrical lens 230 andsubsequently clipped along the nonexpanded dimension by slit shapedaperture 240. The slit width of this aperture 240 is used to control thesheet thickness under the objective (140, shown in FIG. 1), and byextension the depth of field of the microscope. Note that as usedherein, the term “objective” is used in its generic sense to refer tothe last optical component prior to the sample 145. It could be, forexample, a simple lens element, a microscope-style objective, etc.Optionally, the sliding mirror 220, the third cylindrical lens 230 andthe aperture slit 240 may all be mechanically coupled to one another ona mount 250 that is capable of translating from side to side as a singleunit, to provide adjustability. This adjustability may be motor-drivenor manual and various means of adjustment having an equivalent effectwill be evident to persons skilled in the relevant art. A wide varietyof alternative approaches for generating a sheet of light in which allpositions on the sheet are illuminated simultaneously will be readilyapparent to persons skilled in the relevant arts.

In alternative embodiments, the sheet forming optics 110 shapes thelight from the light source 100 by rapidly scanning a pencil beam oflight so as to create a virtual sheet of light. In these embodiments,the illumination at different positions within the sheet occurs atdifferent instants of time. These embodiments create an oblique linebeyond the objective that is then scanned back and forth across thelateral field of view to form the virtual sheet.

FIG. 2B depicts one approach for implementing this virtual sheetembodiment. A light source (e.g., laser 100) emits light (e.g.,monochromatic, coherent, polarized light), and that light is directedthrough an isotropic beam expander 201, 202 that uniformly magnifies theprofile of the light source. The isotopically expanded beam is thendirected onto a galvanometer mirror 260 that reorients the light andscans it into a sheet. Subsequently, the sheet of light is directedthrough a relay telescope 271, 272 and an aperture slit 280. Thefunction of the relay telescope 271, 272 in this embodiment is toprovide additional magnification if necessary and image the galvanometermirror 260 onto a plane conjugate to the back focal plane of theobjective (140, shown in FIG. 1). And the function of the aperture slit280 is similar to the FIG. 2A embodiment described above. Optionally,the galvanometer mirror 260, the relay telescope 271, 272, and theaperture slit 280 may all be mechanically coupled to each other on amount 250 that is capable of translating from side to side as a singleunit to provide similar adjustability to the FIG. 2A embodiment. A widevariety of alternative approaches for using the scanning to generate avirtual sheet of light will be readily apparent to persons skilled inthe relevant arts.

Returning to FIG. 1, light exiting the sheet forming optics 110 entersthe dichroic beam splitter 120, which separates the shorter wavelengthlight used for excitation of fluorescence from the longer wavelengths oflight emitted by the fluorescent molecules. In this embodiment, thedichroic beam splitter 120 was chosen such that the microscope was setup in long-pass configuration wherein fluorescence is transmittedthrough the dichroic beam splitter. The dichroic beam splitter 120reflects the shorter wavelength excitation light towards the scanningelement 125.

In alternative embodiments (e.g., two-photon embodiments, where theexcitation light has a longer wavelength than the fluorescence) thedichroic beam splitter 120 should be configured to reflect the longerwavelengths and pass the shorter wavelength. In some alternativeembodiments, it is also possible to use a shortpass dichroic beamsplitter wherein the microscope is set up in short-pass configuration.Collected fluorescence from the sample would be reflected off of thedichroic beam splitter 120 in this case.

In the FIG. 1 embodiment, the scanning element 125 may be implementedusing a planar scanning mirror with a single degree of freedom. But inalternative embodiments, a wide variety of alternative approaches forimplementing scanning may be used, including but not limited tonon-planer scanning mirrors, moving prisms, etc. The excitation lightarriving from the dichroic beam splitter 120 at the scanning element 125is reflected and scanned by the scanning element 125, after which itcontinues on towards a first set of optical components (e.g., scan-tubelens telescope 131, 132 and objective 140). The first set of opticalcomponents has a proximal end and a distal end, and the scanning element125 routes the excitation light so that it will pass through the firstset of optical components 131-140 in a proximal to distal direction.

An objective 140 is disposed at the distal end of the first set ofoptical components. After passing through the first set of opticalcomponents 131-140, the excitation light will project into a sample 145that is positioned distally beyond the objective 140. The excitationlight is projected into the sample 145 at an oblique angle, and theposition of the excitation light within the sample 145 varies dependingon an orientation of the scanning element 125.

In some embodiments, the excitation light arrives at the back apertureof the objective 140 off-axis. As described previously by Bouchard et al(2015), this creates an oblique sheet of light 142 through the sample145. Note that in those embodiments that employ an adjustable slidingmount (250 in FIGS. 2A and 2B), and the location of the adjustablesliding mount determines the angle of the oblique light sheet throughthe sample 145. It is also possible to adjust the location of theadjustable sliding mount 250 to accommodate for the various backaperture sizes of different objectives 140.

The oblique sheet of light 142 will excite fluorescence in the sample145. Fluorescent light collected from this sample is then collected bythe same objective 140. The objective 140 and the remainder of the firstset of optical components 131, 132 routes detection light from thesample in a distal to proximal direction back to the scanning element125. A conjugate image of the detection light is formed betweencomponents 131 and 132. The location of this conjugate image planechanges in tandem with the laser light sweeping through the sample 145.

The detection light arriving at the scanning element is rerouted by thescanning element 125 so that the detection light will pass through thesecond set of optical components 149-170 in a proximal to distaldirection. In the illustrated embodiment, the second set of opticalcomponents includes an optional emission filter 149 followed by atelescope 151, 155, an aperture stop 160, and a PME 170. Note that inthe FIG. 1 embodiment, the detection light will pass through thedichroic beam splitter 120 before entering the proximal-most element thesecond set of optical components (i.e., lens 151). A conjugate imageplane exists between lenses 151 and 155. Notably, the scanning element125 descans the incoming detection light. As a result of the descanning,this conjugate image plane remains a stationary, despite the fact thatthe excitation light is scanning different sections of the sample 145 inobject space.

Light from the conjugate image plane then continues on through thedistal lens 155 of the telescope and into the PME 170. The phasemodulating element is positioned in the detection optical path at aposition that is distal to the dichroic beam splitter 120. In somepreferred embodiments, the PME 170 is positioned conjugate to the pupilplane of the objective 140.

In a traditional microscope, the sharpest image of the sample is formedwhen the camera is in focus with the primary lens's focal plane; theresolution at this plane is better than the resolution of the planesabove and below and is characterized by the system's point spreadfunction. The role of the PME 170 is to induce a controlled aberrationinto the system such that the point spread functions of points at,above, and below the focal plane (i.e., at different positions along theZ axis) when measured at the camera 190 are homogenized. This aberrationdegrades the point spread function of the microscope at the focal planebut retains the degraded point spread function over a wide range ofdepths. This, in effect, allows the microscope to image multiple planes(i.e. multiple depths) with the same—albeit reduced—in-plane resolution.The introduction of the PME 170 thus improves the system's resolutionand extends the system's depth of field. The general technique is knownin other fields as wavefront encoding. However, here it is employed toderotate a conjugate oblique image plane.

In some preferred embodiments, the PME is a cubic phase plate. In theseembodiments, the phase profile of the cubic phase plate may be describedby the following equation:

θ(x, y)=2πα(x ³ +y ³)

where x and y are normalized pupil coordinates and a is the tuningparameter generally set to |Ψ/2|, where

Ψ(u ₂ , v ₂ ; dz)=−(1/2λ)*NA ²*(u ₂ ² +v ₂ ²)*dz/n,

where λ is the wavelength of light passing through the phase plate, NAis the numerical aperture of the objective 140, u and v are thenormalized pupil coordinates, n is the index of refraction of animmersion medium underneath the objective, and dz is one half the depthof focus (i.e. the range of depths to be imaged). Additional informationregarding characteristics of the phase plate can be found in S. Quinn etal., “Instantaneous Three-Dimensional Sensing Using Spatial LightModulator Illumination with Extended Depth of Field Imaging,” Opt.Express 21, 16007-16021 (2013).

The phase function of the cubic phase plate can also be expressed as thesum of a set of Zernike polynomials as follows:

${\varnothing \left( {x,y} \right)} = {\frac{\pi \; \alpha}{2}\left( {{2Z_{2}} + {2Z_{3}} + Z_{7} + Z_{8} + Z_{10} - Z_{11}} \right)}$

In one example, a mathematical representation of a cubic phase plate wascreated using FRED Optical Engineering Software and simulated to bebetween two thin lenses. An irradiance spread function of an on-axispoint at the focal plane of one of the lenses was determined. A phasefunction that was calculated with an alpha value of 36 and itscorresponding irradiance point spread function are shown in FIGS. 3A and3B, respectively.

Phase plates are generally manufactured with tight tolerances (+/−0.02λ) and are meant to be used over a specific wavelength range. In someembodiments, this wavelength range would correspond to the emissionspectra of whatever fluorescent indicator is being excited in thesample. Typical ranges for these wavelengths will be between 500 and 700nm. In some embodiments, the system may be configured to swap in one ofa plurality of different phase plates depending on the wavelength of thefluorophore that is being excited in the sample 145.

As explained in S. Quinn et al., “Simultaneous Imaging of NeuralActivity in Three Dimensions.,” Front. Neural Circuits 8, 29 (2014), theactual height of the phase plate may be calculated using the followingequation:

${h\left( {x,y} \right)} = {\left( \frac{{\theta \left( {x,y} \right)}*\pi}{2} \right)\left( \frac{\lambda}{n - 1} \right)}$

Where h is the height of the phase plate and n is the refractive indexof the material composing the phase plate.

Designing phase plates specified to tighter wavelength ranges would alsoreduce the chromatic aberrations resulting from deviations from thedesign wavelength. Multi-color imaging can be performed in theconventional sense by placing an image splitter after the lens followingthe phase plate. Alternatively, it is also possible to perform spectralseparation of emitted fluorescence prior to the phase plate. Byincorporating phase plates into a standard image splitter, each phaseplate can be designed for a narrower spectral range.

In alternative embodiments, alternative equations may be implemented inthe phase plate and/or a different class of phase plate (e.g., alogarithmic phase plate) may be used in place of the cubic phase platedescribed above. In some embodiments, the desired phase profile can beetched into a substrate such as glass (e.g. using lithography).

In some embodiments, a different type of phase modulating element 170may be used in place of the phase plate described above. Examples ofsuch alternative phase modulating element include spatial lightmodulators and deformable mirrors. Note that some of these alternativephase modulating elements are programmable (e.g., SLMs). In those cases,any of the phase profiles described above can be imparted onto theprogrammable phase modulating element.

Optionally, a circular aperture (aperture stop 160) may be placed infront of the PME 170 (i.e., on the proximal side of the PME) to modulatethe numerical aperture of the microscope's detection-side optics.Optionally, a long-pass emission filter 149 may be positioned at anyappropriate location in the second set of optical components (e.g.,between the dichroic beam splitter 120 and lens 151, as depicted in FIG.1, or between lens 155 and the camera 190).

After being modulated by the phase modulating element 170 (e.g., thephase plate), the light is captured using a camera 190. The camera isoptically positioned to capture images formed by the detection lightthat has passed through the second set of optical components. In itssimplest form, the camera 190 is a single lens 192 that converges imagelight onto a 2D camera sensor 195. In alternative embodiments, theillustrated single lens 192 can be replaced with a more complex zoomlens module to provide variable magnification to the system. Optionally,image intensifiers and image splitters may also be added for improvedSNR and spectral separation respectively.

The sensor 195 of the camera 190 captures a plurality of frames, each ofwhich represents a 2D image of the fluorescence emanating from a planewithin the sample 145 at a different point in time.

Because the conjugate image plane that appears between lenses 151 and155 is not perpendicular to the axial axis of the second set of opticalcomponents, when that image reaches the sensor 195 of the camera 190,most portions of the images captured by the sensor 195 will be blurred(i.e., out of focus). The PME 170 alleviates this blurred condition to asignificant extent by extending the depth of field in the detectionpath.

This blurred condition can be improved dramatically by processing the 2Dimages that have been captured by the camera's sensor 195. Morespecifically, each of the images that are captured by the camera'ssensor 195 is stored in memory (e.g., RAM, a hard drive, or an SSD), andthose images can be processed by a suitably programmed processor 199 tocorrect for aberration in each of the images. In some embodiments, thisprocessing corrects for aberration by performing deconvolution of ameasured point spread function. In other embodiments, this thisprocessing corrects for aberration by performing deconvolution of asimulated point spread function. Alternative approaches for correctingthe aberration may also be used. The aberration-corrected images arethen stored in memory.

One suitable approach for processing each of the 2D images that has beencaptured by the camera's sensor 195 is as follows: Initially, we notethat the transverse point spread function (X-Y) of a system with a cubicphase plate is depth dependent. In some embodiments, image restorationmay be performed by storing an empirically determined transverse pointspread function at each depth in a look-up table and performingdeconvolution with either a Weiner filter or iterative deconvolution.Any of a wide variety of techniques for implementing deconvolution maybe used, including but not limited to linear autoregressive, ARMA, andWeiner Filters, Lucy-Richardson deconvolution—Bayesian maximumlikelihood expectation maximization solution, Landweber deconvolution,Wavelet based deconvolution, and Maximum Entropy Based Deconvolution.The approaches disclosed in the following three publications, each ofwhich is incorporated herein by reference, may also be used: S. Quirinat al., “Calcium imaging of neural circuits with extended depth-of-fieldLightsheet Microscopy,” Opt. Lett. 41, 855 (2016); 4. D. C. Andreo, etal., “Master in Photonics Fast Image Restoration in Light-SheetFluorescence Microscopy with Extended Depth of Field Using GPUs,”(2015); 0. Olarte, et al., “Decoupled Illumination Detection in LightSheet Microscopy for Fast Volumetric Imaging,” Optica 2, 702-705 (2015).

FIGS. 4A and 4B show the sectioning effect of the oblique light sheet142 used in SCAPE microscopy. The sheet of light 142 scans back andforth within the sample 145 along the X direction and the cameracaptures an image of the sheet in the Y-Z′ dimension. The sheet in SCAPEprovides sectioning along the X dimension in a manner that is similar tothe way that the light sheet in SPIM light sheet systems providessectioning in the Z dimension. The secondary lobes along the X dimensionof the transverse (X-Y) point spread function are projected onto the Z′axis. Because the transverse profile changes slightly over the range ofdepths, the image acquired by the camera frame will have a spatiallyvarying PSF. Depending upon the design of the PME however, uniformitymay be assumed over a part or all of the image. If complete uniformityis assumed, a single point spread function may be acquired/calculatedwithin the center of the Y-Z′ image and the aberrated image may berestored via non-blind deconvolution techniques.

If the point spread function varies dramatically over the range ofdepths (Z′), then a point spread function may be acquired/calculated ateach depth and more complex deconvolution techniques with spatiallyvarying kernels will be used. Examples of suitable techniques fordealing with variations in the point spread function can be found inLauer, Tod, “Deconvolution with a Spatially-Variant PSF,” AstronomicalTelescopes and Instrumentation, International Society for Optics andPhotonics (2002), which is incorporated herein by reference. Alternativetechniques that will be apparent to persons skilled in the relevant artsmay also be used.

Note that the quadratic shift observed in cubic phase plates are aproperty of higher order anti-symmetric phase masks. Rotationallysymmetric phase masks, such as radial quartic and logarithmic functionsinduce no image artifacts but face a steeper tradeoff between contrastand depth of field, as explained in M. Demenikov et al., “ImageArtifacts in Hybrid Imaging Systems with a Cubic Phase Mask.,” Opt.Express 18, 8207-8212 (2010). Regardless, the implementation of suchphase masks in SCAPE may follow an analogous process and would also usenon-blind deconvolution techniques for image restoration.

Returning to FIG. 1, for any given position of the scanning element 125,a sheet of excitation light 142 is projected into the sample 145 at acorresponding position, and a 2D image of the fluorescence from theilluminated plane 142 within the sample 145 is captured by the camera'ssensor 195. More specifically, fluorescence from the illuminated plane142 will be imaged onto a conjugate image plane between lenses 131 and132, be descanned by the scanning element 125, pass through the dichroicbeam splitter 120 and be imaged onto a stationary conjugate image planebetween lenses 151 and 155. This conjugate image plane is stationary dueto the descanning performed by the scanning element 125. This conjugateimage will then be aberrated by the PME 170 as described above and beimaged onto the sensor 195 of the camera 190.

Each frame will then be sequentially read into memory and deconvolved bythe processor 199 with an empirically determined systempoint-spread-function to obtain a reconstructed (i.e. deblurred) imageof the oblique section. As explained above, movement of the scanningelement 125 causes the sheet of excitation light 142 to move to adifferent location within the sample. As a result, if a 2D image iscaptured corresponding to each of a plurality of positions of thescanning element 125, and if each of those images isreconstructed/deblurred as explained above, the result is a stack ofdeblurred 2-D frames that can be assembled into a 3D volume representinga target volume within the sample 145.

Optionally, a plurality of these 3D volumes may be captured at aplurality of different points in time (e.g., every tenth of a second) inorder to depict changes in the volume being imaged (i.e., the volumewithin the sample) over time.

FIG. 5 depicts an alternative embodiment that is similar to the FIG. 1embodiment, except that a reflective phase modulating element 370 isused in place of the transmissive phase modulating element (i.e., PME170 in the FIG. 1 embodiment). Examples of reflective phase modulatingelements 370 that may be used in these embodiments include reflectivespatial light modulators (SLM) and deformable mirrors (DM), each ofwhich is used to modulate the phase profile of the detectedfluorescence. In these embodiments, the reflective phase modulatingelement 370 is preferably positioned at a plane conjugate to the pupilplane of the primary objective 140. In other regards, this FIG. 5embodiment operates in the same way as the FIG. 1 embodiment describedabove.

FIG. 6 depicts another alternative embodiment that has many similaritiesto the FIG. 1 embodiment, but also has a number of significantdifferences. More specifically, the FIG. 6 embodiment scans apencil-shaped beam through the sample using a scanning element 625 withtwo degrees of freedom (e.g., an X-Y galvanometer scanner). In addition,the FIG. 6 embodiment uses a linear sensor 695 (i.e. a one-dimensionalsensor) instead of the 2D sensor described above in connection with theFIG. 1 embodiment.

The FIG. 6 embodiment includes a light source (e.g., laser 100) thatemits light (e.g., monochromatic, coherent, polarized light), and thislight is directed through beam-shaping optics 610 to improve thecharacteristics of the pencil-shaped beam. In some embodiments, the beamshaping optics 610 comprises an isotropic beam expander to uniformlymagnify the profile of the light source, followed by a circular aperturethat clips off the periphery of the beam. The size of this aperture canbe used to control the beam thickness under the objective 140, and byextension the depth of field of the microscope.

Light exiting the beam-shaping optics 610 enters the dichroic beamsplitter 120, which separates the shorter wavelength light used forexcitation of fluorescence from the longer wavelengths of light emittedby the fluorescent molecules. The operation of this beam splitter 120 issimilar to the operation described above in connection with FIG. 1. Thebeam splitter 120 directs the excitation light towards the scanningelement 625.

The scanning element 625 may be implemented using a planar scanningmirror with two degrees of freedom such as an X-Y galvanometer. But inalternative embodiments, a wide variety of alternative approaches forimplementing 2-axis scanning may be used, including but not limited tonon-planer scanning mirrors, moving prisms, etc. The excitation lightarriving from the dichroic beam splitter 120 at the scanning element 625is reflected and scanned by the scanning element 625, after which itcontinues on through the first set of optical components 131-140. Theoperation of these components 131-140 is similar to the operationdescribed above in connection with FIG. 1.

After passing through the first set of optical components 131-140, theexcitation light will project into a sample 145 that is positioneddistally beyond the objective 140. The excitation light is projectedinto the sample 145 at an oblique angle, and the position of theexcitation light within the sample 145 varies depending on anorientation of the scanning element 625. Note that the operation of thisFIG. 6 embodiment to this point is similar to the operation of the FIG.1 embodiment discussed above, with one significant exception: instead ofprojecting a true or virtual sheet of excitation light into the sample145 at an oblique angle, the FIG. 6 embodiment projects a pencil-shapedbeam of excitation light 642 into the sample 145 at an oblique angle. Bycontrolling the position of the scanning element 625 in both the X and Ydirection, the pencil-shaped beam of excitation light in this FIG. 6embodiment can scan through the same volume within the sample that isscanned by the sheet in the FIG. 1 embodiment.

The oblique beam of light 642 will excite fluorescence in the sample145. Fluorescent light collected from this sample is then collected bythe same objective 140. The objective 140 and the remainder of the firstset of optical components 131, 132 routes detection light from thesample in a distal to proximal direction back to the scanning element625. A conjugate image of the detection light is formed betweencomponents 131 and 132. The location of this conjugate image planechanges in tandem with the laser light sweeping through the sample 145.

The detection light arriving at the scanning element is rerouted by thescanning element 625 so that the detection light will pass through thesecond set of optical components 149-170 in a proximal to distaldirection. In the illustrated embodiment, the second set of opticalcomponents includes an optional emission filter 149 followed by atelescope 151, 155, an aperture stop 160, and a PME 170. Note that inthe FIG. 6 embodiment, the detection light will pass through thedichroic beam splitter 120 before entering the proximal-most element thesecond set of optical components (i.e., lens 151). A conjugate imageplane exists between lenses 151 and 155. Notably, the scanning element625 descans the incoming detection light. As a result of the descanning,this conjugate image plane remains a stationary, despite the fact thatthe excitation light is scanning different sections of the sample 145 inobject space.

Light from the conjugate image plane continues through the distal lens155 of the telescope and into the phase modulating element 170. Thephase modulating element is positioned in the detection optical path ata position that is distal to the dichroic beam splitter 120. In somepreferred embodiments, the PME 170 is positioned conjugate to the pupilplane of the objective 140. The implementation of the PME 170 in thisFIG. 6 embodiment follows the same principles discussed above inconnection with the FIG. 1 embodiment.

Optionally, a circular aperture 160 and/or a long-pass emission filter149 may be included, as described above in connection with the FIG. 1embodiment.

After being modulated by the PME 170, the light is captured using acamera 690. The camera is optically positioned to capture images formedby the detection light that has passed through the second set of opticalcomponents. In its simplest form, the camera 690 in this embodiment is asingle lens 692 that converges image light onto a one-dimensional sensor695 (i.e., a linear array).

At any given position of the scanning element 625, a beam of excitationlight 642 is projected into the sample 145 at a corresponding position,and a linear image of the fluorescence from the illuminated line 642within the sample 145 is captured by the camera's sensor 695. Morespecifically, fluorescence from the illuminated line 642 will be imagedonto a conjugate image plane between lenses 131 and 132, be descanned bythe scanning element 625, pass through the dichroic beam splitter 120and be imaged onto a stationary conjugate image plane between lenses 151and 155. This conjugate image plane is stationary due to the descanningperformed by the scanning element 625. This conjugate image will then beaberrated by the PME 170 as described above and be imaged onto thesensor 695 of the camera 690 as a frame of data.

Each frame of data corresponds to a single line of the fluorescenceemanating from a line 642 within the sample 145 at a different point intime. This line will be composed of a plurality of pixels. In someembodiments, the size of each pixel in the linear sensor 695 is between5 and 10 microns. In alternative embodiments, larger pixels are used, inwhich case there will be less resolution in the depth direction. Inother alternative embodiments, the pixel size remains the same (i.e.,5-10 microns), but adjacent pixels are binned together (e.g., in groupsof 4 or 8) to provide increased sensitivity, albeit with decreasedresolution in the depth direction.

Because movement of the scanning element 625 causes any the beam ofexcitation light 642 to move to a different location within the sample,if a linear image is captured corresponding to each of a plurality ofpositions of the scanning element 625, the result is a bundle of frames(similar to a bundle of toothpicks) that can be assembled by processorand associated memory 199 into a 3D volume representing a target volumewithin the sample 145.

Optionally, a plurality of these 3D volumes may be captured at aplurality of different points in time (e.g., every tenth of a second) inorder to depict changes in the volume being imaged (i.e., the volumewithin the sample) over time.

In this FIG. 6 embodiment, deconvolution is not necessary. But in someembodiments, 1D deconvolution may optionally be implemented to deblureach of the 1D images before those images are assembled into a 3Dvolume. In alternative embodiments, linear filtering techniques may beused instead of 1D deconvolution.

In alternative embodiments, the PME 170 of the FIG. 6 embodiment may bereplaced with a reflective PME, similar to the way that the PME 170 inthe FIG. 1 embodiment was replaced with a reflective PME 370 in the FIG.3 embodiment.

In contrast to those embodiments of SCAPE that implement image rotationusing two objective lenses in the detection arm placed at an angle withrespect to one another, the optical components 149-170 in the detectionarm and the camera 190/690 of the FIGS. 1, 5, and 6 embodiments share asingle optical axis. As a result, almost all the light that passesthrough the telescope 151-155 in the detection arm will continue onwardtowards the camera 190/690. This configuration advantageously avoids thelarge losses that are inherent in many prior art SCAPE designs that usean off-axis camera. One complication with this design is that when thecamera 190/690 and the detection arm share a single optical axis, itintroduces on aberration into the image. But this aberration iscorrected algorithmically as explained above.

In this case, although the conjugate image plane is oblique with respectto the optical axis, the camera 190/690 (aligned along the optical axis,as opposed to an oblique angle) will image the oblique conjugate imageplane as a unidimensionally compressed projection onto the surface ofits sensor 195/695. The PME 170 will aberrate the resulting image, inaddition to extending the depth of field of the image reaching thecamera 190/690. These aberrations are corrected algorithmically in theprocessor 199, e.g., by deconvolution of a measured or simulated pointspread function as explained above.

The FIGS. 1, 5, and 6 embodiments described above can advantageouslysimplify alignment, improve resolution, improve light throughput, andenhance the effective detection numerical aperture (with respect to thetwo objective lens embodiments). Superior signal-to-noise ratio can beachieved due to these improvements. This improved S/N can be leveragedin many ways, including imaging at faster rates with shorter exposuretimes in order to reach higher volumetric frame rates and imaging withlower laser intensity reducing photobleaching and damage caused to thesamples. This configuration may also be able to reduce the cost of thesystem by using a microscope-style objective with a lower NA (e.g.,NA˜0.5) and reducing the total footprint. It may be applied towardsendoscopic implementations of SCAPE for rapid in vivo pathology in humantissue or for light-starved, multi-photon implementations of SCAPE. Inthese implementations, the fluorescence may be excited at very lowprobabilities with lasers in the NIR-IR spectrum (700-1600 nm). As aresult, the fluorescence to be collected is significantly weaker thanfor single photon applications (with lasers from 300-700 nm) and couldbenefit from the improved light collection efficiency of the FIGS. 1, 5,and 6 embodiments.

Notably, the numerical aperture, resolution, and light throughput of theFIGS. 1, 5, and 6 embodiments are all governed by the characteristics ofa single primary objective 140. By changing this lens (e.g., using aturret), it becomes possible to provide the modularity of a benchtopmicroscope (e.g., with a number of standardized magnifications,resolutions, and depths of field) while performing fast, volumetricimaging over a large field of view. In some embodiments, this mayinvolve switching in a different PME 170 (e.g., a different phase plate)in tandem with the switching of the objective, or altering the phaseprofile on a programmable phase modulating element (e.g., a deformablemirror or SLM) to account for the changed objective or an alternativemodification. Finally, in comparison to Olarte's, Tomer's and Quirin'sdecoupled illumination/detection scheme in light sheet microscopy, theSCAPE system's lack of orthogonal beam paths extends the application ofthe technologies to large samples such as rodent cortices.

While the present invention has been disclosed with reference to certainembodiments, numerous modifications, alterations, and changes to thedescribed embodiments are possible without departing from the sphere andscope of the present invention, as defined in the appended claims.Accordingly, it is intended that the present invention not be limited tothe described embodiments, but that it has the full scope defined by thelanguage of the following claims, and equivalents thereof.

1. An imaging apparatus comprising: a first set of optical componentshaving a proximal end and a distal end, wherein the first set of opticalcomponents includes an objective disposed at the distal end of the firstset of optical components; a second set of optical components having aproximal end and a distal end, wherein the second set of opticalcomponents includes a phase modulating element; a scanning element thatis disposed proximally with respect to the proximal end of the first setof optical components and proximally with respect to the proximal end ofthe second set of optical components, wherein the scanning element isarranged to route excitation light arriving at the scanning element sothat the excitation light will pass through the first set of opticalcomponents in a proximal to distal direction and project into a samplethat is positioned distally beyond the objective, wherein the excitationlight is projected into the sample at an oblique angle, and wherein theexcitation light is projected into the sample at a position that variesdepending on an orientation of the scanning element, wherein the firstset of optical components is arranged to route detection light from thesample in a distal to proximal direction back to the scanning element,and wherein the scanning element is also arranged to route the detectionlight so that the detection light will pass through the second set ofoptical components in a proximal to distal direction; and a cameraoptically positioned to capture images formed by the detection lightthat has passed through the second set of optical components, whereinthe phase modulating element induces a controlled aberration so as tohomogenize point spread functions of points at, above, and below a focalplane when measured at the camera.
 2. (canceled)
 3. The apparatus ofclaim 1, wherein the phase modulating element comprises a spatial lightmodulator.
 4. (canceled)
 5. The apparatus of claim 1, wherein theobjective has a pupil plane, and the phase modulating element ispositioned at a plane that is conjugate to the pupil plane of theobjective.
 6. The apparatus of claim 5, further comprising an aperturestop positioned (a) adjacent to the phase modulating element and (b)proximally with respect to the phase modulating element.
 7. Theapparatus of claim 1, wherein the detection light forms a stationaryconjugate image plane between the proximal end and the distal end of thesecond set of optical components.
 8. The apparatus of claim 1, whereinthe light detector array comprises a 2D image sensor, wherein the camerasequentially captures a plurality of images formed by the detectionlight that has passed through the second set of optical components, eachof the plurality of images corresponding to a respective frame of imagedate, and wherein the apparatus further comprises a processor programmedto correct for aberration in the frames of image data.
 9. The apparatusof claim 8, wherein the processor corrects for aberration in the framesof image data by performing deconvolution of a point spread function.10. The apparatus of claim 9, wherein the point spread function iseither a measured point spread function or a simulated point spreadfunction. 11-14. (canceled)
 15. The apparatus of claim 1, wherein theexcitation light comprises a sheet of excitation light.
 16. (canceled)17. The apparatus of claim 1, further comprising: a beam splitterdisposed between the proximal end of the second set of opticalcomponents and the scanning element; and a source of the excitationlight, wherein the source of the excitation light is aimed so theexcitation light is directed into the beam splitter, wherein the beamsplitter is arranged to route the excitation light towards the scanningelement, wherein the beam splitter is arranged to route detection lightarriving from the scanning element into the proximal end of the secondset of optical components, wherein the light detector array comprises a2D image sensor, wherein the excitation light comprises a sheet ofexcitation light, wherein the camera sequentially captures a pluralityof images formed by the detection light that has passed through thesecond set of optical components, each of the plurality of imagescorresponding to a respective frame of image date, and wherein theapparatus further comprises a processor programmed to correct foraberration in the frames of image data.
 18. The apparatus of claim 17,wherein the processor corrects for aberration in the frames of imagedata by performing deconvolution of a point spread function.
 19. Theapparatus of claim 18, wherein the objective has a pupil plane, and thephase modulating element is positioned at a plane that is conjugate tothe pupil plane of the objective.
 20. (canceled)
 21. The apparatus ofclaim 1, wherein the phase modulating element has a phase profile ofθ(x, y)=2πα(x ³ +y ³) where x and y are normalized pupil coordinates anda is a tuning parameter set to |Ψ/2|, whereΨ(u ₂ , v ₂ ; dz)=−(1/2λ)*NA ²*(u ₂ ² +v ₂ ²)*dz/n, where λ is awavelength of light passing through the phase modulating element, NA isa numerical aperture of the objective, u and v are normalized pupilcoordinates, n is an index of refraction of an immersion mediumunderneath the objective, and dz is one half of a range of depths to beimaged.
 22. An imaging apparatus comprising: an objective; a pluralityof optical elements disposed in a detection path arranged to receivelight from the objective, wherein the plurality of optical elementsdisposed in the detection path includes a scanning element, wherein thescanning element is arranged to (a) route excitation light into theobjective so as to generate a sweeping excitation beam through a forwardimage plane of the objective and (b) simultaneously route image lightreturning through the objective along the detection path to form aconjugate image; a light detector array positioned to capture images ofthe conjugate image; and a phase modulating element disposed in thedetection path between the scanning element and the light detectorarray, wherein the phase modulating element extends a depth of field inthe detection path.
 23. The apparatus of claim 22, wherein the phasemodulating element extends the depth of field by inducing a controlledaberration so as to homogenize point spread functions of points at,above, and below a focal plane when measured at the light detectorarray.
 24. (canceled)
 25. The apparatus of claim 23, wherein theaberration is corrected by deconvolution of a measured or simulatedpoint spread function.
 26. The apparatus of claim 25, wherein the lightdetector array comprises a 2D image sensor.
 27. The apparatus of claim22, wherein the light detector array comprises a linear image sensor.28. An imaging apparatus comprising: a light source; a cylindrical lensor a scanner that expands light from the light source into a sheet oflight; a beam splitter disposed in a path of the sheet of light; ascanning element disposed in a path of the sheet of light; a firsttelescope having a proximal end and a distal end, with an objectivedisposed at the distal end of the first telescope; a second telescopehaving a proximal end and a distal end, the second telescope having anoptical axis, wherein the beam splitter routes the sheet of lighttowards the scanning element, wherein the scanning element routes thesheet of light into the proximal end of the first telescope, wherein thefirst telescope routes the sheet of light in a proximal to distaldirection through the objective, accepts fluorescent light through theobjective and routes the fluorescent light in a distal to proximaldirection back to the scanning element, wherein the scanning elementroutes the fluorescent light through the beam splitter and into theproximal end of the second telescope, and wherein the second telescopeforms an image from the fluorescent light at a conjugate image plane; acamera positioned on the same optical axis as the second telescope andconfigured to capture blurred images of the conjugate image plane; aphase modulating element disposed between the second telescope and thecamera; and an image processor programmed to deblur images captured bythe camera. 29-30. (canceled)
 31. The apparatus of claim 28 wherein thephase modulating element comprises at least one of a spatial lightmodulator and a deformable mirror. 32-37. (canceled)